Recombinant HIV-1 Gag protein can assemble into virus-like particles (VLPs) in a defined system in vitro. However, under standard conditions, these VLPs have a far smaller radius of curvature than authentic HIV-1 particles. Addition of inositol pentakisphosphate (IP5) to the assembly reaction leads to the formation of VLPs of the correct size. Binding studies with mutant and chimeric proteins suggest that both matrix (MA) and nucleocapsid (NC) domains interact with IP5, despite the flexibility of regions between them. Our research in this area is aimed at identifying the binding sites and testing the possibility that Gag is folded in solution in IP5 unless nucleic acid is present.Role of Inositol Phosphates in HIV-1 Particle Assembly in Living Cells[unreadable] We are investigating whether IP5-related compounds modulate HIV-1 particle assembly in vivo, as well as in vitro. These experiments are being carried out both in cultured human cells and in yeast, using a variety of reagents and techniques that disrupt the metabolism of IP5 derivatives.Analysis of Small Oligomers of HIV-1 Gag[unreadable] Gag oligomerization may be a crucial first step in virus assembly. We found that HIV-1 Gag is in monomer-dimer equilbrium in 0.5 M NaCl. It also appears to oligomerize on short oligonucleotides, perhaps mimicking an early step in assembly. We are testing mutations at possible sites of Gag-Gag interaction for effects on dimerization. Search for Sites of Contact Between HIV-1 Gag Molecules in Assembly[unreadable] To participate in VLP assembly, each Gag molecule must interact with several others. Evidence in the literature suggests that all the relevant sites of Gag-Gag interaction are within the C-terminal domain (CTD) of capsid (CA) and/or in the p2 region between CA and NC. We are testing this idea by analyzing properties of mutants in this region, including their ability to inhibit virion assembly by wild-type Gag. We are also attempting to identify regions of Gag that are protected from hydrogen-deuterium exchange as a result of assembly into VLPs. Structural Analysis of Cylinders Assembled in Vivo from MLV Gag[unreadable] Retrovirus particles are pleomorphic and lack overall icosahedral symmetry. We identified conditions under which the Moloney murine leukemia virus (MLV) Gag protein assembles into cylindrical particles in vivo. Cryo-electron microscopic images of these cylinders exhibit periodicity; we are using this regularity to derive a moderate-resolution structure of MLV Gag protein.Studies on MLV and HIV-1 Maturation[unreadable] The conserved proline at the N-terminus of retroviral CA proteins seems important in the infectious process. MLV mutant proteins in which the N-terminus cannot be cleaved from p12, or in which the N-terminus is glycine rather than proline, are potent trans-dominant inhibitors of wild-type function. To gain insight into the role of proline, we are comparing a series of mutants at the N-terminus of HIV-1 CA with respect to their ability to inhibit the function of wild-type protein, and we are analyzing the functional defects in these mutants and in the mixed particles.[